An ampholytic compound dissolved in an aqueous buffer can be present in the solution either as a cationic, isoelectric or anionic species, depending on the pH of the solution. In the presence of a nonalternating electric field, the cationic or anionic species migrate electrophoretically toward the cathode or the anode, respectively. At a certain solution pH where the ampholyte becomes isoelectric, the net electrophoretic migration of its band becomes zero. The numeric value of this solution pH is equal to the pI value (isoelectric point) of the ampholyte. The pI value of an ampholyte is a material characteristic that is determined by the structure of the ampholyte (the types and pKa values of its weak and strong electrolyte functional groups). When the ampholyte contains a single acidic group (characterized by pKaacid) and a single basic group (characterized by pKaconjugate acid for this conjugate acid form), the pI value of the ampholyte can be calculated as pI=(pKaacid+pKaconjugate acid)/2. When an ampholyte contains two acidic groups (characterized by pKaacid1 and pKaacid2) and one basic group (characterized by pKaconjugate acid for its conjugate acid form) with pKaacid1<pKaacid2<<pKaconjugate acid, the pI of the ampholyte lies between pKaacid1 and pKaacid2 and is calculated as pI=(pKaacid1 pKaacid2)/2. In such cases, the acidic groups between which the pI lies are called the buffering groups, while the basic group is called the titrating group or charge balancing group. Similarly, when an ampholyte contains two basic groups (characterized by pKaconjugate acid1 and pKaconjugate acid2 for their conjugate acid forms) and one acidic group (characterized by pKaacid) with pKaacid<<pKaconjugate acid1<pKaconjugate acid2, the pI of the ampholyte lies between pKaconjugate acid1 and pKaconjugate acid2 and is calculated as pI=(pKaconjugate acid1, pKaconjugate acid2)/2. In such cases, the two basic groups between which the pI lies are called the buffering groups, while the acidic group is called the titrating group or charge balancing group. When the ampholyte contains multiple buffering groups and multiple titrating or charge balancing groups with relatively close pKa values, the charge contribution of each species needs to be evaluated in order to calculate the pI value. A general discussion of ampholytes, isoelectric points, pH gradients and isoelectric focusing can be found in the texts by Righetti, P. G., Isoelectric Focusing: Theory, Methodology and Applications; in Wood, T. S., Burdon, R. H., Eds.; Laboratory Techniques in Biochemistry and Molecular Biology 11; Elsevier Scientific: Amsterdam, 1983; Hjerten, S. in Capillary Electrophoresis: Theory and Practice; Grossman, P. D., Colburn, J. C., Eds.; Academic Press: San Diego, 1992; pp. 191-214; and Giddings, J. C., Unified Separation Science, Wiley-Interscience, New York, 1991, pp 180-182.
Ampholytes are often used in isoelectric focusing (IEF) or isoelectric trapping (IET) separations. Ampholytes whose presence can be detected due to their having a characteristic property—such as UV absorbance or fluorescence or radioactivity—that is different from the properties of the surrounding molecules can be used as pI markers in analytical and preparative-scale IEF separations. pI markers are used for the characterization of the pH gradient that effects the separation of ampholytic analytes. Once the shape of the pH gradient is known, one can determine the pI values of the separated analytes from their focusing position in the pH gradient. (For an excellent discussion of the characteristics and current availability of pI markers see M. Stastna, M. Travnicek and K. Slais, Electrophoresis, 2005, 26, 53-59.) Presently, among others, proteins, peptides, low-molecular weight aminophenols and azo dyes are used as pI markers. The main drawbacks of protein, oligopeptide and peptide pI markers include poor chemical (hydrolytic) stability and inadequate purity, often inadequate UV absorbance and, frequently, lack of fluorescence that can be excited by visible light. Certain peptide pI markers that lack visible light-excited fluorescence and are hydrolytically labile in low pH and high pH solutions are commercially available from Beckman-Coulter (250 South Kraemer Blvd, Brea, Calif. 92821-6232, USA). Fluorescently labeled oligopeptides have been used as pI markers (e.g., Shimura, K., Kasai, K., Electrophoresis 1995, 16, 1479-1484 and Shimura, K., Kamiya, K., Matsumoto, H., Kasai, K, Anal. Chem. 2002, 74, 1046-1053), but they still suffer of hydrolytic instability.
Therefore, synthetic, pure and stable low molecular weight, UV-absorbing and fluorescent pI markers are more desirable than pI markers that are proteins, oligopeptides or peptides. As an additional benefit, the structures of the low molecular weight synthetic pI markers are completely different from those of the separated proteins. Therefore, they are less likely to interfere with the post-IEF use or analysis of the separated protein fractions.
Certain low-molecular weight, UV absorbing pI markers are commercially available (e.g., from Bio-Rad Laboratories, 1000 Alfred Nobel Drive, Hercules, Calif. 94547, USA). The synthesis of other low-molecular weight, UV absorbing pI markers has been described (e.g., see the Stastna reference above). Certain low molecular weight fluorescent pI markers are commercially available (Fluka, Busch, Switzerland) and their use for capillary isoelectric focusing was described (Horka, M., Willimann, T., Blum, M., Nording, P., Friedl, Z., Slais, K., J. Chromatogr. A 2001, 916, 65-71). Unfortunately, their fluorescence had to be excited below 400 nm, outside of the wavelength range of visible light.
Undesirably, the known pI markers often have widely varying structural properties, variable (and often poor) aqueous solubilities (high octanol−water partition coefficients or log Pow values); their pI values cover only certain segments of the needed 3<pI<10 range; the pKa values of the functional groups that straddle the pI value on the acidic and basic sides and are closest to it (pKaclosest) are often farther away from the pI value than 2 (i.e., |pI−pKaclosest|>2), or, expressed in another way, the difference between the closest pKa values that straddle the pI value, ΔpKa=(pKajust above pI−pKajust below pI)>4, the maximum values that still yield adequately (though not greatly) focusing ampholytes (H. Svensson, Acta Chem. Scand. 1962, 16, 456-466). The effective charge of the ampholyte as a function of pH, z(pH), is a material characteristic of the ampholyte. If an ampholyte is to be a rapidly focusing one, it must have a [−dz(pH)/d(pH)] value in the vicinity of its isoelectric point, known as [−dz(pH)/d(pH)pI], that is as large as possible (Giddings, J. C., Unified Separation Science, Wiley-Interscience, New York, 1991, pp 180-182). According to Rilbe (Rilbe, H., Ann. N.Y. Acad. Sci. 209 (1973) 11), [−dz(pH)/d(pH)pI]=ln 10/[1+0.5(10(pKa2-pKa1)/2)] and has a minimum required value of 0.045 in order to focus acceptably. Thus, in order to be an outstanding ampholyte, the pKa values that straddle the pI value need to be as close as possible. A few of the known non-peptide pI markers have [−dz(pH)/d(pH)pI] values in the 0.1 to 0.8 range (Slais, K., Friedl, Z., J. Chromatogr. A 661 (1994) 249-256, and Horka, M., Willimann, T., Blum, M., Nording, P., Friedl, Z., Slais, K., J. Chromatogr. A 916 (2001) 65-71), but very few have [−dz(pH)/d(pH)pI] values above 0.9 (Slais, K., Friedl, Z., J. Chromatogr. A 695 (1995) 113-122).
Thus, there is still an unfilled need for families of nonpeptide ampholytes covering a wide range of pI values with [−dz(pH)/d(pH)pI] values greater than 0.9.
Additionally, up to now, compounds used as pI markers had to be selected by extensive, time consuming trial and error searches (e.g., Righetti, P. G., Gianazza, E., J. Chromatogr. 1977, 137, 171-181), because there were no known correlations between the structure of an ampholyte and its pI value. For example, the respective pKa values of pyridine-2-carboxylic acid (picolinic acid) are 1 and 5.21 yielding pI=3.105 and a |pI−pKaclosest|=2.1; those of pyridine-3-carboxylic acid (nicotinic acid) are 2.07 and 4.66 yielding a pI=3.365 and a |pI−pKacloset|=1.3; while those of pyridine-4-carboxylic acid (isonicotinic acid) are 1.8 and 4.88 yielding a pI of 3.34 and a |pI−pKacloset|=1.54. The pKa values are often significantly different even when two identical functional groups are connected to the same core molecule: for example, the pKa values of the two carboxylic acid groups in 1,2-benzenedicarboxylic acid are 2.76 and 4.92, those in 1,4-benzenedicarboxylic acid are 3.60 and 4.50.
Thus, there is still an unfulfilled need for one or more families of small molecule nonpeptide pI markers that have pI values in the 3<pI<10 range, are characterized by small |pI−pKaclosest| values, small ΔpKa=(pKajust above pI−pKajust below pI) values and high [−−dz(pH)/d(pH)pI] values assuring rapid focusing, have strong UV absorbance, have adequate fluorescence that can be excited by visible light (preferably by the commonly used, commercially available lasers or light emitting diodes, LEDs), have pH-independent fluorescence properties, have adequate water solubility and preferably, have a common core structure that controls light absorbance and fluorescence, and have substituents attached to the core that control the pI value of the ampholyte without altering the light absorption and fluorescence properties of the core. The present invention seeks to fulfill this need and provides further related advantages.